Method and system for wide-field multi-photon microscopy having a confocal excitation plane

Abstract

A wide field microscope includes a stage configured to hold a specimen having a fluorescent material therein, and a multi-photon excitation light source configured to produce excitation light having a single photon energy less than an absorption energy required for single photon excitation of said fluorescent material. A beam expansion unit is optically coupled to the light source and configured to expand the excitation light with reduced pulse spreading characteristics, and an infinity corrected objective optically coupled to the expansion unit and configured to focus the excitation light onto the specimen such that multi-photon excitation of the fluorescent material simultaneously occurs over a predetermined area of the specimen. A focus lens is configured to focus emission light emitted from said predetermined area of the specimen onto at least two pixels of an image detector simultaneously.

1 . A wide-field microscope comprising:
 a stage configured to hold a specimen having a fluorescent material therein;   a multi-photon excitation light source configured to produce excitation light having a single photon energy less than an absorption energy required for single photon excitation of said fluorescent material;   a beam expansion unit optically coupled to the light source and configured to expand the excitation light with reduced pulse spreading characteristics;   an infinity corrected objective optically coupled to the expansion unit and configured to focus the excitation light onto the specimen such that multi-photon excitation of the fluorescent material simultaneously occurs over a predetermined area of the specimen; and   a focus lens configured to focus emission light emitted from said predetermined area of the specimen onto at least two pixels of an image detector simultaneously.   
   
   
       2 . The wide filed microscope of  claim 1 , wherein the beam expansion unit is configured to provide a converging expanded beam of excitation light. 
   
   
       3 . The wide-field microscope of  claim 2 , wherein said microscope is not configured to scan the converging expanded beam of excitation light in an x or y direction across the specimen, the wide-field microscope further comprising a dichroic mirror configured to reflect the excitation light toward the infinity corrected objective lens and to pass the emission light through the dichroic mirror toward the focus lens. 
   
   
       4 . The wide-field microscope of  claim 3 , wherein the dichroic mirror comprises a surface configured to reflect relatively long wavelength excitation light and pass therethrough shorter wavelength emission light, the wide-field microscope of  claim 1 , further comprising a movement system configured to adjust the distance between the infinity corrected objective lens and the specimen held on the stage 
   
   
       5 . The wide-field microscope of  claim 1 , wherein
 said multi-photon excitation light source comprises a pulsed laser light source configured to provide a picosecond, femtosecond, or shorter pulse duration, and   the beam expansion unit is configured to provide substantially no pulse spreading of the pulsed laser light.   
   
   
       6 . The wide-field microscope of  claim 1 , wherein:
 said multi-photon excitation light source comprises a pulsed laser light source configured to provide a picosecond, femtosecond, or shorter pulse duration, and   the beam expansion unit is configured to provide an expanded pulsed laser beam having substantially uniform characteristics across an area of the expanded pulsed laser beam.   
   
   
       7 . The wide-field microscope of  claim 1 , wherein said beam expansion unit is configured to provide a converging expanded beam of excitation light with reduced attenuation characteristics. 
   
   
       8 . The wide-field microscope of  claim 1 , wherein the focusing unit is configured to present a substantially equal amount of optical medium to all light of the expanded beam. 
   
   
       9 . The wide-field microscope of  claim 1 , wherein the laser comprises a tunable laser tunable between approximately 700 nm and approximately 1100 nm. 
   
   
       10 . The wide-field microscope of  claim 1 , wherein the infinity corrected objective comprises:
 a front lens portion configured to focus a beam; and   a rear lens portion configured to maintain a beam substantially parallel.   
   
   
       11 . The wide-field microscope of  claim 1 , wherein the infinity corrected objective lens device comprises an objective having a magnification power between approximately 4 to 100 and a numerical aperture (NA) between approximately 0.10 10 1.4. 
   
   
       12 . The wide-field microscope of  claim 1 , wherein the multi-photon excitation light source is configured to produce excitation light having a photon energy that causes excitation of the specimen only when 2 or more photons are substantially simultaneously absorbed by the fluorescent material. 
   
   
       13 . The wide-field microscope of  claim 17 , wherein the multi-photon excitation light source is configured to produce excitation light having a photon energy that causes excitation of the specimen only when 3 or more photons are substantially simultaneously absorbed by the fluorescent material. 
   
   
       14 . The wide-field microscope of  claim 1 , wherein said beam expansion unit comprises at least one positive mirror configured to expand the excitation light and at least one negative mirror, configured to converge the expanded excitation light into a converging beam. 
   
   
       15 . The wide-field miscroscope of  claim 1 , wherein said beam expansion unit comprises at least one positive mirror configured to expand the excitation light and at least one negative mirror configured to provide a parallel expanded beam from said expanded excitation light. 
   
   
       16 . The wide-field miscroscope of  claim 1  wherein said beam expansion unit comprises at least one concave lens and at least one convex lens, said lenses configured to converge the excitation light into a converging expanded beam of excitation light. 
   
   
       17 . A wide-field microscope comprising:
 means for holding a specimen having a fluorescent material therein;   means for producing excitation light having a single photon energy less than an absorption energy required for single photon excitation of said fluorescent material;   means for expanding the excitation light with reduced pulse spreading characteristics;   means for focusing the excitation light onto the specimen such that multi-photon excitation of the fluorescent material simultaneously occurs over a predetermined area of the specimen; and   means for focusing emission light emitted from said predetermined area of the specimen onto at least two pixels of an image detector simultaneously.   
   
   
       18 . The wide-field microscope of  claim 17 , further comprising means for reflecting the excitation light toward the means for focusing the excitation light and passing the emission light to the means for focusing the emission light. 
   
   
       19 . The wide-field microscope of  claim 18 , further comprising means for filtering the emission light, the means for filtering being disposed in the optical path between the means for reflecting and means for expanding the excitation light. 
   
   
       20 . The wide-field microscope of  claim 18 , further comprising means for moving the specimen relative to the means for expanding the excitation light. 
   
   
       21 . The wide-field microscope of  claim 18 , further comprising means for detecting an image focused on the image plane. 
   
   
       22 . The wide-field microscope of  claim 21 , further comprising means for combining multiple detected images into a three dimensional image of the specimen. 
   
   
       23 . A wide-field microscope comprising:
 a stage configured to hold a specimen having a fluorescent material therein;   a multi-photon excitation light source configured to produce excitation light having a single photon energy less than an absorption energy required for single photon excitation of said fluorescent material;   an optical coupling system optically coupled to the light source and configured to couple the excitation light;   a reflective infinity corrected objective optically coupled to the optical coupling system and configured to focus the excitation light onto the specimen such that multi-photon excitation of the fluorescent material simultaneously occurs over a predetermined area of the specimen; and   a focus lens configured to focus emission light emitted from said predetermined area of the specimen onto at least two pixels of an image detector simultaneously.   
   
   
       24 . The wide-field microscope of  claim 23 , wherein the reflective infinity corrected objective comprises a reflective Schwarzchild microscope objective.